The quest to identify the mechanism underlying adrenergic regulation of cardiac Ca<sup>2+</sup> channels

Roybal, Daniel; Hennessey, Jessica A.; Marx, Steven O.

doi:10.1080/19336950.2020.1740502

Cited by 12 publications

(9 citation statements)

References 74 publications

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“…Phosphorylation is an essential and ubiquitous mode of functional regulation and subcellular localization for many ion channels including voltage-gated cation channels ( Roybal et al, 2020 ), ligand-gated channels ( Nakamura et al, 2015 ), and CFTR, the ion channel whose dysfunction causes cystic fibrosis ( Rowe et al, 2005 ). The functional consequence of phosphorylation ranges from subtle changes in gating equilibria to (in the case of CFTR) a strict dependence on phosphorylation for activity ( Cheng et al, 1991 ).…”

Section: Introductionmentioning

confidence: 99%

Real-time observation of functional specialization among phosphorylation sites in CFTR

Infield

Schene

Fazan

et al. 2023

Journal of General Physiology

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Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from <10% to >1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.

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Section: Introductionmentioning

confidence: 99%

Real-time observation of functional specialization among phosphorylation sites in CFTR

Infield

Schene

Fazan

et al. 2023

Journal of General Physiology

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“…Understanding the molecular mechanisms linking the β-adrenergic signaling cascade to Ca V 1.2 activity promises to shed light on the pathophysiology of chronic heart failure, supraventricular arrhythmias, and other cardiac disorders associated with autonomic dysfunction. However, the molecular mechanisms through which β-adrenergic regulation, increased cAMP, and PKA phosphorylation enhance the activity of Ca V 1.2 channels have remained elusive ( 5 – 8 ).…”

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confidence: 99%

“…Multiple isoforms of Ca V 1.2 channels have been identified in the heart ( 5 – 8 ). The carboxyl-terminal domain (CT) of the pore-forming α 1 subunit mediates interactions between Ca V 1.2 channels that regulate channel activity ( 9 ).…”

mentioning

confidence: 99%

“…The carboxyl-terminal domain (CT) of the pore-forming α 1 subunit mediates interactions between Ca V 1.2 channels that regulate channel activity ( 9 ). Deletion of the distal carboxyl terminus (dCT) increases Ca V 1.2 activity ( 10 ), and up to 80% of the pore-forming α 1 subunits of Ca V 1.2 expressed in the heart are proteolytically processed at position 1800 (Ca V 1.2Δ1800) ( 5 – 8 , 11 , 12 ). The dCT associates noncovalently with the channel, reducing calcium conductance by the Ca V 1.2Δ1800-dCT complex relative to the truncated Ca V 1.2Δ1800 or the full-length channel (Ca V 1.2-FL) ( 12 , 13 ).…”

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confidence: 99%

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Convergent regulation of Ca _V 1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

Hovey

El-Din

Catterall

2022

Proc. Natl. Acad. Sci. U.S.A.

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The L-type calcium currents conducted by the cardiac Ca V 1.2 calcium channel initiate excitation–contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. Ca V 1.2 is regulated by β-adrenergic/protein kinase A (PKA)-mediated protein phosphorylation, proteolytic processing, and autoinhibition by its carboxyl-terminal domain (CT). The small guanosine triphosphatase (GTPase) RAD (Ras associated with diabetes) has emerged as a potent inhibitor of Ca V 1.2, and accumulating evidence suggests a key role for RAD in mediating β-adrenergic/PKA upregulation of channel activity. However, the relative roles of direct phosphorylation of Ca V 1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate Ca V 1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced Ca V 1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased Ca V 1.2 currents. Our findings reveal that the proteolytically processed form of Ca V 1.2 undergoes convergent regulation by direct phosphorylation of the CT and by phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for upregulation of the activity of Ca V 1.2 channels in the fight-or-flight response.

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“…However, numerous studies critically challenged this theory. In particular, mutated Ca V 1.2 channels in genetically engineered mice lacking putative PKA phosphorylation sites on α 1C and/or β 2b , were still upregulated by PKA 7, 13–16 (reviewed in 4, 17 ).…”

Section: Introductionmentioning

confidence: 99%

Reconstitution of β-adrenergic regulation of Ca_V1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

Katz

Subramaniam

Chomsky-Hecht

et al. 2020

Preprint

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Introduction: Cardiac L-type voltage-gated CaV1.2 channels are crucial in physiological regulation of cardiac excitation-contraction coupling. Adrenergic modulation of CaV1.2 starts with activation of β-adrenergic receptors (AR) and culminates in protein kinase A (PKA) - induced increase of calcium influx through CaV1.2 channels. To date, this cascade has never been fully reconstituted in heterologous systems; even partial reconstitution proved challenging and controversial. A recent study identified Rad, a calcium channel inhibitory protein, as an essential component of the adrenergic signaling cascade. We corroborated this finding, further characterized, and fully reconstituted, the complete β-AR CaV1.2 modulation cascade in a heterologous expression system. Objective: Our primary goal was to heterologously reconstitute the complete β-adrenergic cascade, and to investigate the role of Rad and additional molecular determinants in adrenergic regulation of cardiac CaV1.2. Methods and Results: We utilized the Xenopus oocyte heterologous expression system. We expressed CaV1.2 channel subunits, without or with Rad and β1-AR or β2-AR. To activate PKA, we injected cyclic AMP (cAMP) into the oocytes, or extracellularly applied isoproterenol (Iso) to stimulate β-AR. Whole-cell Ba2+ currents served as readout. We find and distinguish between two distinct pathways of PKA modulation of CaV1.2: Rad-dependent (~80% of total) and Rad-independent. We separate the two mechanisms by showing distinct requirements for the cytosolic N- and distal C- termini of α1C and for the CaVβ subunit. Finally, for the first time, we reconstitute the complete pathway using agonist activation of either β1-AR or β2-AR. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes, such as a >2-fold increase in CaV1.2 current, a hyperpolarizing shift in activation curve, and a high constitutive activity of β2-AR. Conclusions: The adrenergic modulation of CaV1.2 is composed of two distinct pathways, Rad-independent and Rad-dependent. The latter contributes most of the β-AR-induced enhancement of CaV1.2 activity, crucially depends on CaVβ subunit, and is differently regulated by β1-AR and β2-AR. The reconstitution of the full β-AR cascade provides the means to address central unresolved issues related to roles of auxiliary proteins in the cascade, CaV1.2 isoforms, and will help to develop therapies for catecholamine-induced cardiac pathologies.

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The quest to identify the mechanism underlying adrenergic regulation of cardiac Ca²⁺ channels

Cited by 12 publications

References 74 publications

Real-time observation of functional specialization among phosphorylation sites in CFTR

Real-time observation of functional specialization among phosphorylation sites in CFTR

Convergent regulation of Ca _V 1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

Reconstitution of β-adrenergic regulation of Ca_V1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

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The quest to identify the mechanism underlying adrenergic regulation of cardiac Ca2+ channels

Cited by 12 publications

References 74 publications

Real-time observation of functional specialization among phosphorylation sites in CFTR

Real-time observation of functional specialization among phosphorylation sites in CFTR

Convergent regulation of Ca V 1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

Reconstitution of β-adrenergic regulation of CaV1.2: Rad-dependent and Rad-independent protein kinase A mechanisms

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The quest to identify the mechanism underlying adrenergic regulation of cardiac Ca²⁺ channels

Convergent regulation of Ca _V 1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

Reconstitution of β-adrenergic regulation of Ca_V1.2: Rad-dependent and Rad-independent protein kinase A mechanisms